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Background research:

Proteins are essential nutrients for a large class of organisms, especially homo sapiens or
humans. Proteins exist in food we eat. Foods such as fish, potato and egg has a large amount
of proteins. Proteins in our body can not be absorbed if it is not broken down into its building
blocks, Amino acids. Amino acids are linked to each other by peptide bond through the process
of condensation reaction by ribosomes to form polypeptides. Most commonly there are twenty
different types of amino acids which ribosomes uses to make polypeptides. The difference
between amino acids is in its R group. Any combination of these amino acids is possible and
this gives a huge diverse types of polypeptides. Genes in the DNA gives the code for the
sequence of amino acids. Since there are a variety of genes in DNA a lot of different
polypeptides can be formed. This is the shape of the protein which determines its function in the
cells.Three dimensional shapes of proteins are determined by sequence of amino acids and
polypeptides. Proteins such as globular proteins fold up as they are made and take a specific
three dimensional shape. The final conformation of proteins are achieved by bonds or
interaction between R groups of amino acids within the polypeptides. These bonds are weak
and can be broken down easily which changes the conformation of protein and is called
denaturation. Normally the denaturation is permanent. Enzymes such as pepsin are proteins.
Thousands of metabolic reactions in our body occurring all the time during our life at a very fast
rate is impossible to occur without the help of enzymes. In other words its enzymes which brings
the activation energy of reactions low and catalysis the chemical reactions in our body.
Enzymes like pepsin have a special region called active site. Substrates such as albumin of egg
white binds with this region of pepsin and an altar in the shape of enzyme help the breakdown
of the substrate without break of the enzyme itself. Each enzyme has specific active site and
therefore binds with specific substrates and responsible for specific reaction. Temperature and
pH can alter the shape of the enzyme and consequently the active site and this denaturation
causes the rate of reaction to slow down and eventually stop. (Allott and Mindorff)
Research question: How pH buffer of 2, 3, 4, 5, 6 and 7 affects the reaction of pepsin and eggwhite?
Purpose: To look that enzyme pepsin reacts well in lower pH buffer zones and increase in ph
slows down the rate of reaction and at last stops it.
Hypothesis:
Pepsin was identified for the first time by Theodor Schwann a German physiologist in 1836.
Pepsin is in gastric juices of stomach of most mammals. Pepsin do not digest the glands of
stomach membrane that produces them although the cells itself are mostly made of proteins.
This is because when they are produced they are in inactive form called pepsinogen.
Pepsinogen in acidic environment of stomach unfolds, changes to pepsin and starts breaking
the proteins in the stomach. This reaction is called hydrolysis which is the reverse reaction of
condensation. In hydrolysis reaction a water molecules is needed to break up the peptide bonds
of polypeptide. The split causes the hydrogen atom of water to bind with one amino acid and the
OH group with the other amino acid. This is shown in diagram 1.1. Enzymes work most
efficiently within a specific temperature and pH which is called optimum temperature or pH. The

optimum temperature for pepsin is 37-40 degree celsius and the optimum pH is 1.5-2.5 ("Pepsin
| Biochemistry"). Therefore It is hypothesized that in low ph e.g. 2 and 3 pepsin reacts well with
albumin than higher pHs. This will be tested by measuring the weight lost of egg-white pieces
after staying of 24 hours in solution of buffer and pepsin.
Diagrams:
Figure 1.1

Hydrolysis and condensation reaction.


Independent variable (the variable that we change in our experiment) : pH buffer of 2, 3, 4, 5,
6 and 7.
Dependent variable (the one that you will measure): The amount of weight lost by the eggwhite pieces measured in grams.
Constants:
- Temperature: the temperature will be kept at 37 degree celsius. This is normal
body temperature for humans and pepsin digest egg in our stomach at this temperature.
- Amount of pepsin:
- Amount of pH buffer:

Materials:
1. 10 Boiled eggs (protein albumin)
2. 1 knife

3. Beakers (1 100 ml and 1 2000 ml)


4. 1 electronic balance of uncertainty of 0.01 gram
5. 30 test tubes with test tube rack
6. laptop
7. pH buffer of (2, 3, 4, 5, 6 and 7)
8. 1 pipette
9. 2 grams of pepsin enzyme powder.
10. White strip for labeling the test tubes.
11. Marker
12. Heater
13. Thermometer
14. 500ml of water
15. Distilled water

Procedure:
1. Label the test tubes using white strip for each pH buffer and trial as 21 , 22 . 25 ,
31, 35 for five trails and pHs of (2, 3, 4, 5, 6 and 7). Bigger digit indicating the pH and
the smaller one the trial.
2. peel the boiled eggs and separate the white part from yolk.
3. cut the white part into smaller pieces
4. Weigh the white small pieces using electronic balance and record the data in
laptop
5. Put each piece in test tube after weighing
6. Make a 1% solution of pepsin enzyme by mixing 2 grams of pepsin with 50ml of
water(distilled)
7. Using a graduated pipette put 2 ml of 1% pepsin solution to each test tube
8. Put 2 ml of each of buffer solutions into each test tubes according to lebel using a
pipette
9. Shake the test tubes gently so that the buffer and enzymes mixes
10. Make a water bath in a 2000 ml beaker of temperature 37 C. Have the beaker
about less than half full.
11. Put a thermometer into the beaker and beaker on the heater.
12. Place all the test tubes in the water bath and leave it for 24 hours
13. After 24 hours put the test tubes back in the rack
14. Pour a way the solution of the test tubes
15. Soak the egg pieces and weight them again
16. Record the weights in your laptop according to the labels of each test tube.

Bibliography:
- Allott, Andrew and David Mindorff. Biology. 2014. Print.
- "Pepsin | Biochemistry". Encyclopedia Britannica. N.p., 2015. Web. 17 Sept.
2016.